This study was
conducted to determine the effects of different levels of dietary vitamin C on
the growth performance and body composition of Salmo trutta caspius fry (mean initial weight of 100±10mg) for 6 weeks
experimental period. Five purified diets contained 0, 100, 500, 1000 and 2000 mg ascorbic acid (LAPP) per kg diet were
formulated The results showed that the growth performance of fish was affected
by the ascorbic acid content of different diets. The highest survival, body
weight gain and SGR were obtained from diet four (1000 mg
LAPP per kg diet). No significant difference was observed in the whole-body
proximate composition of S. trutta caspius but fat content showed a significant difference. These findings
suggest that vitamin C could be added as a dietary supplement for improving physiological
performance and optimal growth of S. trutta caspius.

Keywords; Salmo trutta caspius; Vitamin C; Survival;
Body Composition

Vitamin C plays an
important role in growth and immunity of fish (Lin and Shiau, 2005). It is the
most important water-soluble antioxidant and performs as a co-factor in many
hydroxylating reactions (Kumari and sahoo, 2005). The most of teleost fish are unable
to synthesize ascorbic acid due to the lack of the last enzyme of the
biosynthetic pathway: L-gulonolactone oxidase. Therefore, Vitamin C must be
supplied via the feed to obtain optimal growth, immunity, reproduction and
survival. (Verlhac, 1999; Dobrowski, 2001). The studies also have been shown
importance of relationship between vitamin C and properties such as growth,
survival and body composition in aquatic animals. (Ai et al, 2006; Bae et al,
2012; Xiao et al 2010; Mai et al, 1998; Falahatkar et al, 2006; Misra et al,
2007) There are a few studies on the nutrition requirements of S. trutta caspius but no information is reported on its
requirement of vitamin C. The aim of present study is to determine the effects
of different levels of dietary vitamin C on survival, growth and body
composition of this fish.

Material
and Methods

At the beginning a
batch of Caspian brown trout eggs were obtained from the breeding and
cultivation center of Gezel Cheshme Kowsar (Tonekabon, Iran). The eggs were
transferred to the hatchery of marine sciences faculty of Tarbiat Modares
University (Noor, Mazandaran, Iran) and incubated in a rectangular trough with
recirculation water. After yolk sac absorption and start of exogenous feeding,
some fries were choosing for the experiment. Fries were reared in five groups
contained different levels of vitamin C (as mentioned in below) for 6 weeks.
Each group was stocked in 90 liter tank in triplicate and each tank was
contained 70 fish with initial weigh of 100 ± 10 mg. Fry were fed five times
daily and all groups of fish were fed their respective diet by hand to visual
satiety (visual observation of first feed refusal)

The formulation of
experimental diets was performed by Lindo software at concentrations of 0 or
control (D1), 100 (D2), 500 (D3), 1000

Survival Rate (%)=
The number of fish in first period/ the number of fish in final period*100

The proximate
composition of experimental diets and fish whole body were performed using the
standard methods of Association of Official Analytical Chemists (AOAC, 1995).
Dry matter, crude protein, crude lipid and ash were measured by drying at 105 0C
for 24 h, Kjeldahl method, Soxhlet method and combustion at 550 0C,
respectively.

All data were
subjected to One-way ANOVA analysis by using SPSS 17.0. Differences between the
means were determined by Duncan test. The level of significance chosen was
P<0.05 and the results are presented as means ± standard deviation (SD).

Results
and Discussion

Some common
deficiency signs, including scoliosis and lordosis, poor growth, higher
mortality and lower SGR were observed in the fish of the control group (without
vitamin c). The growth performance of fish was affected by the ascorbic acid
content of different diets. As dietary ascorbic acid increased from 0 to 1000
mg kg-1 diet, the SGR increased from 2.55 to 4.06 % day-1
and survival rate increased from 53.33 to 84%. (Table 2). The fish fed the control diet
significantly had a lower survival than other groups. Such results were
reported from other species (Bae et al, 2012; Xiao et al 2010; Misra et al,
2007 and Ai et al, 2006). But in compare with some species which are able to
Vit. C de novo synthesis, results are different. (Falahatkar et al, 2006; Mai,
1998). The metabolic rate is the primary factor regulating the AA requirements.
Therefore, fish larva, displaying relatively a faster growth and metabolism
than juveniles and adults, might need higher dietary AA levels to sustain
optimal growth and physiological condition (Merchie et al, 1997).

No significant
difference was observed in the whole-body proximate composition of S. trutta
caspius but fat amount showed a significant difference in different groups.
Same results on body composition were obtained in other studies (Bae et al,
2012; Xiao et al 2010). Dietary vitamins can affect on metabolism and body
composition(Hwang and Lim,2002). The results showed that fat level was high in
D4 and it could be due to more metabolism in fat synthesis. These findings were
observed in other report (Falahatkar et al, 2006 ).

The present study
was intended to determine the effects of different levels of dietary vitamin C
on two antioxidant enzymes including superoxide dismutase (SOD) and catalase
(CAT) from Salmo trutta
caspius fry during 6
weeks. Five purified diets contained 0, 100, 500, 1000 and 2000 mg ascorbic
acid (LAPP) per kg diet were formulated for mentioned study. The findings
showed that the CAT activity was significantly affected by the ascorbic acid
content of different diets, while no significant difference was observed in SOD
activity among dietary vitamin C treats. The highest activity of both enzymes
was obtained from diet without vitamin C that reveals fry is in oxidative
condition and more sensitive.

2003). Vitamin C is
an important dietary antioxidant, it significantly decreases the adverse effect
of free radicals such as reactive oxygen and nitrogen species that can cause
oxidative damage to macromolecules such as lipids, DNA and proteins. In
addition, ascorbic acid can regenerate other antioxidants such as a-tocopheroxyl, urate and P-carotene
radical cation from their radical species. Thus, ascorbic acid acts as
co-antioxidant for a-tocopherol by converting a-tocopheroxyl radical to a-tocopherol
and helps to prevent the a-tocopheroxyl
radical mediated peroxidation reactions (Nuida, 2003). Several studies have
examined the influence of nutritional variations on antioxidant enzymes in fish
(Lygren., et al 2000; Tocher., 2002; Mourente., 2002; Puangkaew et al 2005).
The aim of this study was to examine the impact of dietary vitamin C
supplementation on SOD and CAT activity.

Material
and Methods

At the beginning a
batch of Caspian brown trout eggs were obtained from the breeding and
cultivation center of Gezel Cheshme Kowsar (Tonekabon, Iran). The eggs were
transferred to the hatchery of marine sciences faculty of Tarbiat Modares
University (Noor, Mazandaran, Iran) and incubated in a rectangular trough with
recirculation water. After yolk sac absorption and start of exogenous feeding,
some fries were choosing for the experiment. Fries were reared in five groups
contained different levels of vitamin C (as mentioned in below) for 6 weeks.
Each group was stocked in 90 liter tank in triplicate and each tank was
contained 70 fish with initial weigh of 100 ± 10 mg. Fry were fed five times
daily. The formulation of experimental diets was performed by Lindo software at
concentrations of 0 (or control; D1), 100 (D2), 500 (D3), 1000 (D4) and 2000 (D5)
mg vitamin C (LAPP) Kg-1
diet according to (Table
1).

End of the
experiment amount of 4 fish from each replicate were sampled for the analysis.
The muscle were homogenized (1:10, w/v) in homogenization buffer containing 100
mM potassium phosphate buffer (pH 7.4), 100 mM KCl and 1 mM EDTA for 1.5 min.
Homogenates were centrifuged at 10,000 g for 30 min (+4 ° C). Supernatants were
used as enzyme source (Atli and Canli, 2010).

The activity of Superoxide dismutase (SOD, EC 1.15.1.1) was assayed based on the ability of SOD to
inhibit the reduction of NBT (Nitroblue tetrazolium) by superoxide anion
(Winterbourn et al, 1975; Worthington Enzyme Manual 1993). One unit is defined
as the amount of enzyme causing half the maximum inhibition of NBT reduction.
Different volumes of extracts were added to cuvettes containing 0.2 mL of 0.1M
EDTA, 0.1 mL of 1.5 mM NBT and reached to 3ml with 67 mM phosphate buffer (pH
7.8). Then, 0.05 mL of 0.12 mM riboflavin was added at zero time and at timed
intervals. All cuvettes were incubated in a light box for 12 min and absorbance
at 560 nm was read at timed intervals by a spectrophotometer (PerkinElmer,
Lambda25; USA). The amount of enzyme resulting in 50% of maximum inhibition of
NBT reduction was determined. Catalase activity (CAT, EC 1.15.1.1) was determined according to Aebi (1984) by
following the decrease in absorbance at 240 nm due to hydrogen peroxide (H2O2) consumption. The difference in absorbance
per unit time (60 s in our assay) was used as a measure of CAT activity. Total
soluble protein was measured by

Mean values of
enzyme activities were compared among the different groups using one-way
analysis of variance (ANOVA). Differences between the means were determined by
Duncan test. The level of significance chosen was P<0.05 and the results are
presented as means ± standard deviation (SD).

Result
and Discussion

Catalase activities
were significantly decreased in S. trutta caspiuss fed with diet C5 compared with control group and no significant
difference was observed in SOD activity among dietary vitamin C treats but SOD
activity was lower in diets with higher amount of vitamin C (Table 2). Similary
Mourente et al., 2002 repoted the both of catalase and SOD activities at Sparus aurata were reduced by dietary vitamin E as
antioxidant after 30 days feeding.

In another study,
the level of dietary vitamin E showed some significant effects on the
activities of the enzymes of the liver antioxidant defense system of juvenile
turbot (Scophthalmus
maximu.), halibut (Hippoglossus hippoglossus) and sea bream (Sparus aurata) (Tocher et tal., 2002). No interactions
were observed between dietary vitamin E and antioxidant enzyme activities in
atlantic salmon but liver SOD and GPX activities were significantly reduced in
hyperoxygenated fish although catalase activity was unaffected (Lygren et al.
2000). CAT is a scavenger of H2O2, therefore its high activity in control group
could indicate the presence of a large amount of H2O2 in the system (Puangkaew
et al., 2005). Decreased production and non-availability of the substrate (O2-) in response to antioxidant may be a reason
for decreased SOD activities. (Janssens et al., 2000).